Compositions comprising immunoreactive reagents and saponins, and methods of use thereof

ABSTRACT

The present invention relates to pharmaceutical compositions that are useful for the prevention and treatment of infectious diseases, primary and metastatic neoplastic diseases (i.e., cancer), neurodegenerative or amyloid diseases, or any other disease wherein the treatment of such disease would be improved by an enhanced immune response, and methods of formulating the compositions. The compositions comprise an immunoreactive reagent (i.e., an antigen binding protein comprising an antigen binding region and a region or regions of an antibody that mediate antibody dependent immunological processes) and a saponin. The present invention also relates to methods of using the compositions of the invention for the prevention and/or treatment of infectious diseases, primary and metastatic neoplastic diseases (i.e., cancer), neurodegenerative or amyloid diseases, or any other disease wherein the treatment of such disease would be improved by an enhanced immune response.

1. FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions that areuseful for the prevention and treatment of infectious diseases, primaryand metastatic neoplastic diseases (i.e., cancer), neurodegenerative oramyloid diseases, or any other disease wherein the treatment of suchdisease would be improved by an enhanced immune response, and methods offormulating the compositions. The compositions comprise animmunoreactive reagent (i.e., an antigen binding protein comprising anantigen binding region and a region or regions of an antibody thatmediate antibody dependent immunological processes) and a saponin. Suchantibody dependent processes include, but are not limited to, antibodydependent cellular cytotoxicity and phagocytosis. The present inventionalso relates to methods of using the compositions of the invention forthe treatment of infectious diseases, primary and metastatic neoplasticdiseases (i.e., cancer), neurodegenerative or amyloid diseases, or anyother disease wherein the treatment of such disease would be improved byan enhanced immune response. Such methods include passive immunotherapy(i.e., passive immunization with an immunoreactive reagent, such as anantibody). Any such passive immunotherapy may be enhanced by theco-administration of a saponin, preferably QS-21.

2. BACKGROUND OF THE INVENTION

2.1. Passive Immunotherapy

Passive immunotherapy (also termed passive immunization) refers to theadministration of an immunoreactive reagent (i.e., an antibody)comprising, for example, an antigen binding region directed against anepitope on a pathogen, tumor or pathogenic protein, and an Fcreceptor-binding region, directly to a patient. The immunoreactivereagent can be given prophylactically to, for example, inhibitinfection, or therapeutically to reduce or eliminate infection, toreduce or eliminate cancer cells, or to clear or remove pathogenicproteins, e.g., protein aggregates or deposits, as occurs inneurodegenerative and/or amyloidogenic disease. This is distinguishedfrom immunization of a patient with a protein to induce an in vivoimmune response to produce antibodies. Such administration preferablyresults in the stimulation of effector cells with Fc receptors capableof interacting with the Fc portion (i.e., the Fc receptor bindingregion) of the antibody or immunoreactive agent, resulting in cellularimmune functions such as antibody-dependent cellular cytotoxicity (e.g.,ADCC) or antibody-mediated opsonization and/or phagocytosis directedagainst the cell, pathogen, or protein possessing the epitope recognizedby the immunoreactive agent. The saponin-mediated enhancement of passiveimmunotherapy can occur through stimulation of effector cells, i.e.,induction and/or activiation of the Fc receptors on such cells. Theefficacy of antibody-mediated tumor therapy which depends on FcReffector cell functions can be modified by the use of specificcytokines. Keler, et al., 2000, J. Immunol. 164:5746-5752.

2.2. Saponins

Quillaja saponins are a mixture of triterpene glycosides extracted fromthe bark of the tree Quillaja saponaria. They have long been recognizedas immune stimulators that can be used as vaccine adjuvants, (Campbelland Peerbaye, 1992, Res. Immunol. 143(5):526-530), and a number ofcommercially available complex saponin extracts have been utilized asadjuvants. Crude saponins have been extensively employed as adjuvants inveterinary vaccines against foot and mouth disease, and in amplifyingthe protective immunity conferred by experimental vaccines againstprotozoal parasites such as malaria, Trypanosoma cruzi plasmodium, andthe humoral response to sheep red blood cells (SRBC) (Bomford, 1982,Int. Arch. Allerg Appl. Immun. 67:127).

The first commercially available Quillaja saponin adjuvants were crudeextracts which, because of their variability, were not desirable for usein veterinary practice or in pharmaceutical compositions for man. Anearly attempt to purify Quillaja saponin adjuvants was made by Dalsgaard(1974, Archiv fuer die gesamte Virusforschung 44:243). Dalsgaardpartially purified an aqueous extract of the saponin adjuvant materialfrom Quillaja saponaria Molina. However, while Dalsgaard's preparation,“Quil-A,” was a definite improvement over the previously availablecommercial saponins, it still exhibited considerable heterogeneity.

Subsequent analysis via high-pressure liquid chromatography showed thatQuil A was in fact a heterogeneous mixture of structurally relatedtriterpene glycosides (U.S. Pat. No. 5,057,540; Kersten et al., 1988,Infect. Immun. 56:432-438; Kensil et al., 1991, J. Immunol. 146:431-437;Kensil et al., 1991, J. Am. Vet. Med. Assoc. 199:1423-1427). However,not all of these saponins were active as adjuvants.

The four most predominant purified Quillaja saponins are QS-7, QS-17,QS-18, and QS-21 (alternatively identified as QA-7, QA-17, QA-18, andQA-21). These saponins have been purified by HPLC and low pressuresilica chromatography and were found to be adjuvant active, althoughdiffering in biological activities such as hemolysis and toxicity inmice. In particular, QS-21 and QS-7 were found to be least toxic in mice(Kensil et al., 1991, J. Immunol. 146:431-437).

Due to its potent adjuvant activity and low toxicity, QS-21(commercially available as the “Stimulon®” adjuvant) has been identifiedas a useful immunological adjuvant (Kensil et al., 1995, “Structural andImmunological Characterization of the Vaccine Adjuvant QS-21,” inVaccine Design: The Subunit and Adjuvant Approach, Powell and Newmaneds., Plenum Press, New York). QS-21 is a complex triterpene glycosideof quillaic acid. QS-21 is glycosylated at triterpene carbon 3,triterpene carbon 28, and carbon 5 of the second fatty acyl unit in afatty acid domain.

More recently, QS-21 was further purified using hydrophilic interactionchromatography (HILIC) and resolved into two peaks, QS-21-V1 andQS-21-V2, which have been shown to be chemically different compounds. InC57BL/6 mice immunized with vaccines consisting of ovalbumin and eitherQS-21, QS-1-V1, or QS-21-V2, both of the individual components QS-21-V1and QS-21-V2 are comparable in adjuvant effect to the original QS-21peak (containing a mixture of 3:2 QS-21-V1 and QS-21-V2) for boostingthe IgG subclasses IgG1, IgG2b, and IgG2 as well as the total IgG titer(U.S. Pat. No. 5,583,112, the entire contents of which are herebyincorporated by reference). Quillaja saponins are structurally distinctfrom the saponins derived from other plant species. Two structuralfeatures that distinguish Quillaja saponaria saponins from those ofother plant species are a fatty acid domain and a triterpene aldehyde atcarbon 4 of the triterpene. (Kensil et al., 1995, “Structural andImmunological Characterization of the Vaccine Adjuvant QS-21,” inVaccine Design: The Subunit and Adjuvant Approach, Powell and Newmaneds., Plenum Press, New York). Modifications to the aldehyde on thetriterpene indicate that this functional group may be involved in theadjuvant mechanism (Soltysik et al., 1995, Vaccine 13(15):1403-1410).

Quillaja saponins, particularly QS-7, QS-17, QS-18, and QS-21, have beenfound to be excellent stimulators of antibody response to solubleT-dependent protein antigens, “subunit antigens,” which are poorlyimmunogenic and require a potent adjuvant for maximization of immuneresponses. Examples of purified subunit antigens for which saponinadjuvants that augment the IgG response in mice include keyhole limpethemocyanin (KLH), HIV-1 gp120 (Bomford et al., 1992, AIDS Res. Hum.Retroviruses 8:1765), and influenza nucleoprotein (Brett et al., 1993,Immunology 80:306). QS-7, QS-17, QS-18, and QS-21 have also been shownto stimulate potent antibody responses in mice to the antigens bovineserum albumin and cytochrome b₅ (Kensil et al., 1991, J. Immunol.146:431). The level of antibody response induced by these purifiedsaponins was comparable to other commonly used adjuvants, e.g., completeFreund's adjuvant, and superior to aluminum hydroxide.

QS-21 has also been shown to enhance antibody responses to T-independentantigens, including unconjugated bacterial polysaccharides (White etal., 1991, “A purified saponin acts as an adjuvant for a T-independentantigen,” in: Immunobiology of Proteins and Peptides, Vol. VI (Atassied.), Plenum Press, New York, pp. 207-210). The immunogenicity of thevaccine was further increased by conjugating diphtheria toxoid to thepolysaccharide. QS-21 enhanced the antibody response to thepolysaccharide as well as the carrier, including IgG2a, IgG2b, and IgG3responses (Coughlin et al., 1995, Vaccine 13(1):17-21).

The ability of adjuvants to modulate the isotype distribution and IgGsubclass distribution of antibody response to an antigen through thepromotion of Ig subclass switching has important implications forimmunity to many bacterial and viral vaccines. QS-7, QS-17, QS-18, andQS-21 stimulate IgG2a response to cytochrome b5 after administrationwith saponin doses of 20 μg (Kensil et al., 1991, J. Immunol 146:431).In this regard, QS-21 shifts predominant IgG1 responses to a profilethat includes significant IgG2b and IgG2a responses. For example, QS-21has been shown to stimulate antigen-specific IgG2a to a number ofantigens, including Borrelia burgdorferi outer surface proteins OspA andOspB (Ma et al., 1994, Vaccine 12(10):925), feline leukemia virus (FeLV)envelope gp70 (Kensil et al., 1991, J. Am. Vet. Med. Assoc. 10:1423),human cytomegalovirus (HCMV) envelope protein gB (Britt et al., 1995, J.Infect. Dis. 171:18), respiratory synctial virus (RSV) purified fusionprotein (Hancock et al., 1995, Vaccine 13(4):391), and tetanus toxoid(Coughlin et al., 1995, Vaccine 13(1):17). QS-21 has also been shown toinduce boostable antibody responses (Britt et al., 1995, J. Infect. Dis.171:18-25; Helling et al., 1995, Cancer Res. 55:2783-2788).

The ability of the QS-21 adjuvant to induce class I majorhistocompatibility complex (MC) antigen-restricted cytotoxicT-lymphocyte responses (CTL) after immunization with soluble proteins isa characteristic of saponin adjuvants. A number of studies have shownthe ability of QS-21 to induce potent cytotoxic T-lymphocyte (CTL)responses to various antigens, including ovalbumin (Wu et al., 1994,Cell. Immunol. 154:394-406; Newman et al., 1992, J. Immunol148(8):2357-2362), recombinant HIV-1 gp160 protein (Wu et al., 1992, J.Immunol. 148:1519), respiratory syncytial virus (“RSV”) purified fusionprotein (Hancock et al., 1995, Vaccine 13(4):391), and subunitSIV_(mac251) gag and env (Newman et al., 1994, AIDS Res. Hum.Retroviruses 10(7):853).

Most of the saponin adjuvant studies have been carried out in mice.However, the adjuvant activity of saponins is not limited to mice; ithas also been demonstrated in humans, cats, dogs, guinea pigs, rabbits,pigs, sheep, cattle, and nonhuman primates. (Kensil et al., 1995,“Structural and Immunological Characterization of the Vaccine AdjuvantQS-21,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell, M.F. and Newman, M. J. eds., Plenuim Press, New York).

Phase 1 human trials of QS-21 with GM2 ganglioside-keyhole limpethaemocyanin conjugate vaccine have been conducted in patients withmalignant melanoma (Livingston et al., 1994, Vaccine 12:1275-1280.Increased immunogenicity after administration with QS-21 adjuvant wasobserved (Helling et al., 1995, Cancer Res. 55:2783-2788). In anotherset of clinical trials, QS-21 was found to be a potent immunologicaladjuvant that significantly increased the serological response ofmelanoma patients to the murine anti-idiotype antibody MELIMMUNE-1(Livingston et al., 1995, Vaccine Res. 4(2):87).

The immune adjuvant effect of saponins is dependent upon dose. Dependingupon the antigen and the species, a minimum dose level of QS-21 isrequired for optimum response (Kensil et al., 1991, J. Immunol.146(2):431-7; Kensil et al., 1993, Ann NY Acad Sci. 690:392-5; Newman etal., 1992, J. Immunol. 148(5):1519-25; Livingston et al., 1994, Vaccine12(14):1275-80). Below this minimum dose, the immune adjuvant effect issuboptimal (either low level or absent). QS-7 also has a dose responsecurve (Kensil et al., 1991, J. Immunol. 146(2):431-7).

Saponins have also been discovered to elicit an innate immune responsewhich is independent of any particular antigen. The innate immunitystimulated by saponins results in a potentiated immune system that iscapable of responding to an immunological challenge in an enhancedmanner. For example, saponins are capable of increasing the productionof TNF-alpha, IL-6 and MIP-1-alpha in macrophage cells. In bone marrowderived dendritic cells, saponins increase the production of MIP-1-alphaand IL-1, decrease the production of E-12 and MIP-1-beta. This effect ofsaponins is described in International Patent Publication-No. WO01/51083, incorporated herein in its entirety. This property of saponinsis different from their adjuvant effects in that an adjuvant effect isspecific to the particular antigen with which the adjuvant isadministered, while the innate immunity stimulation effect results in ageneral enhancement of the immune system and its ability to respond to achallenge which is independent of the particular antigen used tochallenge. Measurements of innate immunity, and methods of determiningenhancement thereof are known in the art, and are described inInternational Patent Publication No. WO 01/51083.

3. SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions that areuseful for the prevention and treatment of infectious diseases, primaryand metastatic neoplastic diseases (i.e., cancer), neurodegenerative oramyloid diseases, or any other disease wherein the treatment of suchdisease would be improved by an enhanced immune response, and methods offormulating the compositions. The compositions comprise animmunoreactive reagent (i.e., an antigen binding protein comprising anantigen binding region and a region that mediates one or more antibodydependent immunological processes) and a saponin. The compositions mayfurther comprise an immunostimulatory oligonucleotide.

The present invention also relates to methods of using the compositionsof the invention for the treatment of infectious diseases, primary andmetastatic neoplastic diseases (i.e., cancer), neurodegenerative oramyloid diseases, or any other treatment of a disease that would beimproved by an enhanced immune response. Such methods include passiveimmunotherapy. Any such passive immumotherapy may be enhanced by theco-administration of a saponin, preferably QS-21. The saponin may beadministered concurrently with the immunoreactive reagent, or within ashort time either before or after the administration of theimmunoreactive reagent. A short time includes, but is not limited to 1,15 or 30 minutes, 1, 6 or 12 hours, or 1 or 2 days. In a preferredembodiment, the saponin is QS-7, QS-17, QS-18, QS-21, QS-21-V1, orQS-21-V2.

The compositions of the invention can be used to generate an immuneresponse against epitopes associated with neurodegenerative or amyloiddiseases, cancer or an agent of infectious disease or any cell ormolecule bearing an epitope associated with the aforementioned diseases,by administering to an individual a therapeutic amount of theimmunoreactive reagent and saponin. Where an immune response against atype of cancer is desired, an immunoreactive reagent is used whichspecifically binds to (or “recognizes”) an antigen of the type ofcancer, i.e., a tumor-associated antigen. Where eliciting an immuneresponse against an agent of an infectious disease is desired, animmunoreactive reagent is used which specifically binds to an antigen orpathologic protein (i.e., toxin) of the agent of infectious disease. Inother embodiments, the compositions of the invention that comprise animmunoreactive reagent that specifically binds to an antigen of a typeof cancer are used to treat or prevent the type of cancer; and thecompositions of the invention that comprise an immunoreactive reagentthat specifically binds to an agent of an infectious disease are used totreat or prevent the infectious disease. In other embodiments, thecompositions of the invention comprise an immunoreactive reagent thatspecifically binds an antigenic molecule associated with aneurodegenerative disease or an amyloid disease ane are used to treat orprevent said neurodegenerative or amyloid disease.

Prophylactic and therapeutic dosage regimens and kits are also providedby the invention.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions that areuseful for the prevention and treatment of infectious diseases, primaryand metastatic neoplastic diseases (i.e., cancer), neurodegenerative oramyloid diseases, or any other disease wherein the treatment of suchdisease would be improved by an enhanced immune response, and methods offormulating the compositions. In certain embodiments, the outcome ofeliciting an immune response is prophylaxis or therapy. The compositionsof the invention can be used to provide improved passive immunotherapyagainst cancer or an agent of infectious disease orneurodegenerative/amyloid diseases or any other disease or pathologicalstate that can be treated by passive immunotherapy, by administering toan individual a therapeutic amount of the immunoreactive reagent andsaponin.

Cancers that can be treated according to the methods of the inventioninclude, but are not limited to, leukemia (e.g., acute leukemia such asacute lymphocytic leukemia and acute myelocytic leukemia), neoplasms,tumors (e.g., non-Hodgkin's lymphoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma), heavy chain disease (B-cell lymphoma), metastases, orany disease or disorder characterized by uncontrolled cell growth. Tumorantigens or tumor associated antigens include cancer-germ cell (CG)antigens (MAGE, NY-ESO-1), mutational antigens (MUM-1, p53, CDK4),over-expressed self-antigens (p53, HER2/NEU), viral antigens (fromPapilloma Virus, Epstein-Barr Virus), tumor proteins derived fromnon-primary open reading frame mRNA sequences (NY-ESO1, LAGE1), Melan A,MART-1, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, tyrosinase, gp100, gp75,HER-2/neu, c-erb-B2, CEA, PSA, MUC-1, CA-125, Stn, TAG-72, KSA (17-1A),PSMA, p53 (point mutated and/or overexpressed), RAS (point mutated),EGF-R, VEGF, GD2, GM2, GD3, Anti-Id, CD20, CD19, CD22, CD36, Aberrantclass II, B1, CD25 (IL-2R) (anti-TAC), or HPV.

Infectious agents that can be treated according to the inventioninclude, but are not limited to viruses, such as hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral miningitis,encephalitis, dengue or small pox; bacteria, such as mycobacteriarickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi(Lyme disease), Bacillus antracis (anthrax), tetanus, streptococcus,staphylococcus, mycobacterium, tetanus, pertissus, cholera, plague,diptheria, chlamydia, S. aureus and legionella; and agents of protozoaldisease, such as leishmania, kokzidioa, trypanosoma or malaria.

Immunoreactive reagents specifically binding an antigenic molecule in oron a cell or structure, e.g., extracellular deposits or plaquescomprising peptide and/or protein fibrils, that displays the hallmarksof a neurodegenerative or amyloid disease may also be utilized.Preferably, where it is desired to treat or prevent neurodegenerative oramyloid diseases, immunoreactive reagents that specifically bind tomolecules comprising epitopes of antigenic molecules associated withneurodegenerative diseases, or epitopes of antigenic moleculesassociated with amyloid diseases, including but not limited to fibrilpeptides or proteins, are used. Such neurodegenerativedisease-associated antigenic molecules may be molecules associated withAlzheimer's Disease, age-related loss of cognitive function, seniledementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson'sDisease, cerebral palsy, progressive supranuclear palsy, Guam disease,Lewy body dementia, prion diseases, spongiform encephalopathies,Creutzfeldt-Jakob disease, polyglutamine diseases, Huntington's disease,myotonic dystrophy, Freidrich's ataxia, ataxia, Gilles de la Tourette'ssyndrome, seizure disorders, epilepsy, chronic seizure disorder, stroke,brain trauma, spinal cord trauma, AIDS dementia, alcoholism, autism,retinal ischemia, glaucoma, autonomic function disorder, hypertension,neuropsychiatric disorder, schizophrenia, or schizoaffective disorder.Examples of such antigentic molecules are disclosed in U.S. applicationSer. No. 09/489,216, which is incorporated by reference herein in itsentirety, and include, but are not limited to, β-amyloid or a fragmentthereof, an oligomeric Aβ complex or a fragment thereof, an ApoE4-Aβcomplex, tau protein or a fragment thereof, amyloid precursor protein ora fragment thereof, a mutant amyloid precursor protein or a fragmentthereof, presenillin or a fragment thereof, a mutant of presenillin or afragment thereof, α-synuclein or a fragment thereof, or a prion proteinor a fragment thereof, and the antigenic derivatives of any of theforegoing proteins or fragments thereof. Amyloid disease associatedantigenic molecules may be molecules associated with diseasescharacterized by the extracellular deposition of protein and/or peptidefibrils which form amyloid deposits or plaques, including but notlimited to type U diabetes and amyloidoses associated with chronicinflammatory or infectious disease states and malignant neoplasms, e.g.,myeloma. Certain amyloid disease such as Alzheimner's disease and priondiseases, e.g., Creutzfeldt Jacob disease, are neurodegenerativediseases.

The treatment of other disease or pathogenic states that can be treatedby immunotherapy may are also within the scope of the present invention.Such treatment includes the treatment of cardiovascular disease with animmunoreactive reagent that binds angiotensin 2, and the treatment ofautoimmune associated diseases, such as arthritis, with immunoreactivereagents that bind IL-10, tumor necrosis factor, or otherimmunoregulatory molecules.

The compositions comprise an immunoreactive reagent (i.e., an antigenbinding protein comprising an antigen binding region and a region thatmediates one or more antibody dependent immunological processes, e.g.,an Fc receptor-binding region) and a saponin. In certain embodiments,the compositions of the invention further include an immunostimulatoryoligonucleotide.

The present invention also relates to methods of using the compositionsof the invention for the treatment of infectious diseases, primary andmetastatic neoplastic diseases (i.e., cancer), neurodegenerative oramyloid diseases, or any other treatment of a disease that would beimproved by an enhanced immune response. Such methods include passiveimmunotherapy. Any such passive immunotherapy may be enhanced by theco-administration of a saponin, preferably QS-21. The saponin may beadministered concurrently with the immunoreactive reagent, or within ashort time either before or after the administration of theimmunoreactive reagent. A short time includes, but is not limited to 1,15 or 30 minutes, 1, 6, 12, 18 or 36 hours, or 1 or 2 days.

In a preferred embodiment, the saponin is QS-7, QS-21, QS-21-V1, orQS-21-V2. In another embodiment, the saponin is an active derivative,e.g., semi-synthetic derivatives such as GP1-100, as well as otherimmuostimulatory (e.g., adjuvant active) saponins or saponin-containingcompositions. The adjuvant, haemolytic, and innate immune stimulatoryactivities of individual saponins have been extensively studied in theart. (Lacaille-Dubois and Wagner, 1996 “A review of the biological andpharmacological activities of saponins.” Phytomedicine vol. 2, pp363-386, incorporated herein by reference in its entirety). Such activesaponins include Quil A and fractions thereof, ISCOMS, saponins derivedfrom other plant species such as Gypsophila and Saponaria (Bomford etal., 1992, Vaccine 10(9):572-577, incorporated herein by reference inits entirety), and Chenopodium quinoa saponins have been used in bothintranasal and intragastric vaccines (Estrada et al., 1998, Comp.Immunol. Microbiol. Infect. Dis. 21(3):225-36, incorporated herein byreference in its entirety).

The compositions can be utilized for the prevention of a variety ofcancers, e.g., in individuals who are predisposed as a result offamilial history or in individuals with an enhanced risk to cancer dueto environmental factors, for the prevention of infectious diseases,e.g., in individuals with enhanced risks of exposure to agents ofinfectious disease, and for the prevention of neurodegenerative oramyloid diseases, for example in individuals with geneticpredispositions to neurodegenerative or amyloid diseases.

The invention also provides kits comprising one or more containers withone or more of the ingredients of the pharmaceutical compositions of theinvention. Optionally associated with such kit(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In one embodiment, the kits can optionally furthercomprise a predetermined amount of the immunoreative reagent (i.e., anantigen binding protein comprising an antigen binding region and aregion that mediates one or more antibody dependent immunologicalprocesses, e.g., an Fc receptor-binding region) and a saponin. In apreferred embodiment, the kit comprises the immunoreactive reagent andthe saponin in separate containers.

4.1. Immunoreactive Reagents

The immunoreactive reagents of the invention are peptides comprising 1)an antigen binding region and, optionally, 2) a region that mediates oneor more antibody dependent immunological processes. The antigen bindingregion can comprise or consist of the antigen binding region of anantibody. The antigen binding region can comprise any peptide or domainthat interacts specifically with an antigen of interest. For example,the antigen binding region can be a ligand or other specific bindingpartner of the antigen of interest, or can be a fragment of such ligandor binding partner, or can be derived from such ligand or bindingpartner.

The region that mediates one or more antibody dependent immunologicalprocesses can comprise or consist of a region that is capable of bindingan Fc receptor, e.g., the portion of an antibody that binds Fcreceptors, or a region that binds complement, e.g., the complementbinding region of an antibody. This region can also be an antigenbinding domain of an antibody that binds to Fc receptors or complement.

Immunoreactive reagents include antibodies, Fab and F(ab′)₂ fragments,molecules or proteins engineered to include the antigen binding portionof an antibody, molecules or proteins engineered to include an antigenbinding domain that mediates antibody dependent immune responses, apeptide or domain that interacts specifically with the antigen ofinterest, or any antigen binding domain that interacts with anantigen/epitope of interest, and the domain of the constant region of anantibody that mediates antibody dependent immune effector cell responsesor processes. Examples of such domains or regions within the Ab constantregion that can be used in the present invention include those disclosedin Reddy et al., 2000, J. Immunol. 164(4):1925-33; Coloma et al., 1997,Nat Biotechnol. 15(2):159-63; Carayannopoulos et al., 1994, Proc Natl.Acad. Sci. U.S.A. 91(18):8348-52; Morrison, 1992, Annu Recombinantexpression vector Immunol. 10:239-65; Traunecker et al., 1992, Int. J.Cancer Suppl., 7:51-2; Gillies et al., 1990, Hum. Antibodies Hybridomas,1 (1):47-54; each of which is incorporated herein by reference in itsentirety.

Such antibody dependent processes include, but are not limited to,antibody dependent cellular cytotoxicity, activation of complement,opsonization and phagocytosis. The effector cells that mediate certainantibody dependent processes include monocytes, macrophages, naturalkiller cells, and polymorphonuclear cells. Without being bound by aparticular mechanism, it is thought that saponins are able to increasereceptors on the effector cells responsible for mediating the antibodydependent response. These receptors include the Fc alpha and Fc gammareceptors, isoforms thereof, or any combination thereof. Thus, in aparticular embodiment, the region of the immunoreactive reagent thatmediates one or more antibody dependent immunological processescomprises or consists of a region that is a ligand for Fc receptors,preferably the Fc a receptor or the Fc gamma receptor, or both. Inanother embodiment, the region of the immunoreactive reagent thatmediates one or more antibody dependent immunological processescomprises or consists of a region that stimulates the function of immuneeffector cells, preferably monocytes, macrophages, natural killer cells,polymorphonuclear cells, or any combination of two or more of suchcells, such that a prophylactic and/or therapeutic effect is achieved.

In a preferred embodiment, the immunoreactive reagent is an antibody, ora composition comprising an antibody such as serum. In a particularembodiment, the immunoreactive reagent is an IgA, IgG or IgM antibody,or comprises a fragment thereof. In a particularly preferred embodiment,the immunoreactive reagent is a monoclonal antibody, or includesfragments of a monoclonal antibody. The immunoreactive reagent may alsocomprise or consist of human immune globulin for treatment of HepatitisB; Respigam for the treatment of RSV; Sandoglobulin, or ImmuneGlobulinIV (IGIV). In another embodiment, the immunoreactive reagent is notdirected towards any single epitope, but instead comprises a mixture ofone or more molecules that bind to a population of epitopes. An exampleof such an immunoreactive reagent is serum or antibodies concentratedfrom serum or plasma. Such serum or plasma may be from a subjectimmunized against a particular antigen, or from a subject not soimmunized.

In another embodiment, the immunoreactive reagent is a bi-specificmolecule having two antigen binding regions of different specificity,i.e., one recognizing an epitope on a target cell or protein, and theother recognizing an epitope of an effector cell, e.g., an epitope ofFcR. In another embodiment, the immunoreactive reagent is a bi-specificmolecule having two antigen binding domains for different epitopes onthe target cell/protein and a domain that mediates antibody dependentimmune responses. Such bi-specific molecules that target cancer cells orpathogens and their therapeutic effects have been examined both in vivoand in vitro (e.g., Wallace et al., 2001, J Immunol. Methods248(1-2):167-82; Sundarapandiyan et al., 2001, J Immunol. Methods248(1-2):113-23; Honeychurch et al., 2000, Blood 96(10):3544-52; Negriet al., 1995, Br J Cancer 72(4):928-33; Wang et al., 1994, ZhonghuaZhong Liu Za Zhi 16(2):83-7, Chinese) (each of which is incorporatedherein by reference in its entirety).

In a preferred embodiment, the immunoreactive reagent is purified.“Purified” as used herein to describe certain peptides, antibodies,molecules, proteins, antigens, saponins, and the like, refer to a statebeyond that in which the molecules, proteins, antigens, and the like,are separated from greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 99% of the proteins, polysaccharides, and/orlipids with which the peptides, antibodies, molecules, proteins,antigens, saponins, and the like are normally associated naturally. Ifthe isolated molecules, proteins, antigens, saponins, and the like aresynthesized, they are contaminated with less than 50%, 40%, 30%, 20%,10%, 5%, 1% or 0.1% of the chemical precursors or synthesis reagentsused to synthesize the molecules, proteins, antigens, saponins, and thelike. In preferred embodiments the peptides, antibodies, molecules,proteins, antigens, saponins, and the like are at least 1% pure, 5%pure, 10% pure, 20% pure, 30% pure, 40% pure, 50% pure, 60% pure, 70%pure, 80% pure, 90% pure, 95% pure, 99% pure, or 100% pure. As usedherein, the term “% pure” indicates the percentage of the totalcomposition that is made up of the molecule of interest, by weight.Thus, a composition of 100 grams containing 50 grams of a molecule ofinterest is 50% pure with respect to the molecule of interest.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated herein by reference in their entireties). The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CHI region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theimmunoglobulins, or fragments thereof, of the present invention includethose disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough etal., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCTapplication No. PCT/GB91/01134; PCT publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; and Sawaiet al., 1995, AJRI 34:26-34; and Better et al., 1988, Science240:1041-1043 (each of which is incorporated by reference in itsentirety). Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology203:46-88; Shu et al., 1993, PNAS 90:7995-7999; and Skerra et al., 1988,Science 240:1038-1040.

For some uses, including in vivo use of antibodies in humans, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a constantregion derived from a human immunoglobulin. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties. Humanized antibodies are antibody molecules fromnon-human species that bind the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Often,framework residues in the human framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Reichmann et al., 1988, Nature332:323, which are incorporated herein by reference in their entireties.Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; Roguska et al., 1994, Proc Natl. Acad. Sci. USA,91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332), all of whichare hereby incorporated by reference in their entireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced-using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, 1995,Int. Rev. Immunol. 13:65-93. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., 1988, Bio/technology12:899-903).

In a preferred embodiment, the antibodies have in vivo therapeuticand/or prophylactic uses. Examples of therapeutic and prophylacticantibodies include, but are not limited to, SYNAGIS® (MedImmune, MD)which is a humanized anti-respiratory syncytial virus (RSV) monoclonalantibody for the treatment of patients with RSV infection; HERCEPTIN®(Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonalantibody for the treatment of patients with metastatic breast cancer;REMICADE® (infliximab) (Centocor, PA) which is a chimeric anti-TNFαmonoclonal antibody for the treatment of patients with Crone's disease;REOPRO® (abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIareceptor on the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD18 F(ab′)₂ (Genentech); CDP860 which is a humanizedanti-CD 18 F(ab′)₂ (Celltech, UK); PRO542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); Ostavir whichis a human anti Hepatitis B virus antibody (Protein DesignLab/Novartis); PROTOVIR™ which is a humanized anti-CMV IgG1 antibody(Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murineanti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14 which is an anti-CD14antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech);OVAREX™ which is a murine anti-CA 125 antibody (Altarex); PANOREX™ whichis a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);Smart M195 which is a humanized anti-CD33 IgG antibody (Protein DesignLab/Kanebo); RITXANT which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanizedanti-HLA antibody (Protein Design Lab); ONCOLYM1M (Lym-1) is aradiolabelled murine anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone);ABX-IL8 is a human anti-IL8 antibody (Abgenix); anti-CD11a is ahumanized IgG1 antibody (Genentech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(DEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering);SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies,respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tanox Biosystems); ABX-CBL is amurine anti CD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgGantibody (Medimmune/Bio Transplant); Orthoclone/OKT3 is a murineanti-CD3 IgG2a antibody (ortho Biotech); SIMULEC™ is a chimericanti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanizedanti-β₂-integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine antiCD18 F(ab′)₂ (Pasteur-Merieux/Immunotech); CAT-152 is a humananti-TGF-β₂ antibody (Cambridge Ab Tech); and Corsevin M is a chimericanti-Factor VII antibody (Centocor). The above-listed immunoreactivereagents, as well as any other immunoreactive reagents, may beadministered according to any regimen known to those of skill in theart, including the regimens recommended by the suppliers of theimmunoreactive reagents. Immunoreactive reagents that bind to 4-1BBglycoprotein or Cytotoxic T Lymphocyte Antigen-4 (CTLA-4), such asantibodies specific for 4-1BB or CTLA-4, respectively, are alsocontemplated within the present invention.

The immunoreactive reagents of the invention can be produced by anymethod known in the art for the synthesis of antibodies, in particular,by chemical synthesis or preferably, by recombinant expressiontechniques. Such methods are described below with reference to anantibody immunoreactive reagent, but are readily applicable to theproduction of other immunoreactive reagents.

The nucleotide sequence encoding an antibody or other immunoreactivereagent may be obtained from any information available to those of skillin the art (i.e., from Genbank, the literature, or by routine cloning).If a clone containing a nucleic acid encoding a particular antibody oran epitope-binding fragment thereof or other immunoreactive reagent isnot available, but the sequence of the antibody molecule orepitope-binding fragment thereof or other immunoreactive reagent isknown, a nucleic acid encoding the immunoglobulin or otherimmunoreactive reagent may be chemically synthesized or obtained from asuitable source (e.g., an antibody cDNA library, or a cDNA librarygenerated from, or nucleic acid, preferably poly A+ RNA, isolated fromany tissue or cells expressing the antibody, such as hybridoma cellsselected to express an antibody) by PCR amplification using syntheticprimers hybridizable to the 3′ and 5′ ends of the sequence or by cloningusing an oligonucleotide probe specific for the particular gene sequenceto identify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.In the case of immunoreactive reagents that do not exist in nature,nucleic acids encoding the different regions of the immunoreactivereagent can be obtained from preexisting libraries or known genes, orcan be synthesized.

Once the nucleotide sequence of the antibody or other immunoreactivereagent is determined, the nucleotide sequence of the antibody or otherimmunoreactive reagent may be manipulated using methods well known inthe art for the manipulation of nucleotide sequences, e.g., recombinantDNA techniques, site directed mutagenesis, PCR, etc. (see, for example,the techniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; and Ausubel et al., eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY, which are both incorporated byreference herein in their entireties), to generate antibodies or otherimmunoreactive reagent having a different amino acid sequence by, forexample, introducing amino acid substitutions, deletions, and/orinsertions into the epitope-binding domain regions of the antibodies orother immunoreactive reagent or into the constant (Fc) regions of theantibodies or other immunoreactive reagent which are involved in theinteraction with immune effector cells.

Recombinant expression of an antibody or other immunoreactive reagentrequires construction of an expression vector containing a nucleotidesequence that encodes the antibody or other immunoreactive reagent. Oncea nucleotide sequence encoding an antibody molecule or a heavy or lightchain of an antibody, or portion thereof (preferably, but notnecessarily, containing the heavy or light chain variable region) orother immunoreactive reagent has been obtained, the vector for theproduction of the antibody molecule or other immunoreactive reagent maybe produced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody or other immunoreactive reagentencoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or other immunoreactive reagentcoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and id vivo geneticrecombination. The nucleotide sequence encoding the heavy-chain variableor constant region, light-chain variable or constant region, both theheavy-chain and light-chain variable regions, an epitope-bindingfragment of the heavy- and/or light-chain variable region, or one ormore complementarity determining regions (CDRs) of an antibody or otherimmunoreactive reagent may be cloned into such a vector for expression.The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules or other immunoreactive reagent of the invention.Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule orother immunoreactive reagent of the invention in situ. These include,but are not limited to, microorganisms such as bacteria (e.g., E. coliand B. subtilis) transformed with recombinant bacteriophage DNA, plasmidDNA or cosmid DNA expression vectors containing antibody or otherimmunoreactive reagent coding sequences; yeast (e.g., Saccharomyces andPichia) transformed with recombinant yeast expression vectors containingantibody or other immunoreactive reagent coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody or other immunoreactive reagent codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody or other immunoreactive reagent codingsequences; and mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 andNSO cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,bacterial cells such as Escherichia coli, and more preferably,eukaryotic cells, especially for the expression of whole recombinantantibody molecule or other immunoreactive reagent, are used for theexpression of a recombinant antibody or other immunoreactive reagentmolecule. For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for antibodies (Foecking et al., 1986, Gene 45:101,and Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule or other immunoreactive reagent being expressed. For example,when a large quantity of such a protein is to be produced, for thegeneration of pharmaceutical compositions of an antibody molecule,vectors which direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO 12:1791), in which the antibody or otherimmunoreactive reagent coding sequence may be ligated individually intothe vector in frame with the lacZ coding region so that a fusion proteinis produced; and pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109, and Van Heeke & Schuster, 1989, J. Biol. Chem.24:5503-5509).

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody or other immunoreactivereagent coding sequence may be cloned individually into non-essentialregions (for example the polylhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized to express an antibody molecule or other immunoreactivereagent of the invention. In cases where an adenovirus is used as anexpression vector, the antibody or other immunoreactive reagent codingsequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe antibody molecule or other immunoreactive reagent in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).Specific initiation signals may also be required for efficienttranslation of inserted antibody or other immunoreactive reagent codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the antibody or other immunoreactive reagent sequences, ormodifies and processes the antibody or other immunoreactive reagent inthe specific fashion desired. Such modifications (e.g., glycosylation)and processing (e.g. cleavage) of protein products may be important forthe function of the antibody or other immunoreactive reagent. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the antibody or otherimmunoreactive reagent expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and inparticular, myeloma cells such as NSO cells, and related cell lines,see, for example, Morrison et al., U.S. Pat. No. 5,807,715, which ishereby incorporated by reference in its entirety.

For long-term, high-yield production of recombinant antibodies or otherimmunoreactive reagent, stable expression is preferred. For example,cell lines which stably express the antibody molecule or otherimmunoreactive reagents may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule or other immunoreactive reagent. Such engineered cell lines maybe particularly useful in screening and evaluation of compositions thatinteract directly or indirectly with the antibody molecule or otherimmunoreactive reagent.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357, and O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan andAnderson, 1993, Ann. Rev. Biochem. 62: 191-217; and May, 1993, TIB TECH11(5):155-2 15); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; in Chapters 12 and 13, Dracopoliet al. (eds), 1994, Current Protocols in Human Genetics, John Wiley &Sons, NY; and Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, whichare incorporated by reference herein in their entireties.

The expression levels of an antibody molecule or other immunoreactivereagent can be increased by vector amplification (for a review, seeBebbington and Hentschel, 1987, The use of vectors based on geneamplification for the expression of cloned genes in mammalian cells inDNA cloning, Vol. 3. Academic Press, New York). When a marker in thevector system expressing antibody or other immunoreactive reagent is 1amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody or otherimmunoreactive reagent gene, production of the antibody or otherimmunoreactive reagent will also increase (Crouse et al., 1983, Mol.Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc.Natl. Acad. Sci. USA 77:2 197). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

Once an antibody or other immunoreactive reagent molecule of theinvention has been produced by recombinant expression, it may bepurified by any method known in the art for purification of animmunoglobulin molecule or other immunoreactive reagent, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A purification, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard techniques for the purification of proteins. Further, theantibodies or other immunoreactive reagents of the present invention orfragments thereof may be fused to heterologous polypeptide sequencesdescribed herein or otherwise known in the art to facilitatepurification.

4.2. Sources of Saponins

Any saponin or saponin preparation known in the art may be used in thecompositions and methods of the invention. The term “saponin” as usedherein includes glycosidic triterpenoid compounds which produce foam inaqueous solution and have hemolytic activity in most cases. Theinvention encompasses the use of saponins per se, as well as natural andpharmaceutically acceptable salts and pharmaceutically acceptablederivatives thereof. The term “saponin” also embodies biologicallyactive fragments thereof. The term “saponin” also encompasses chemicallymodified saponins, such as GPI-0100, and other modified saponinsdescribed, for example, in U.S. Pat. Nos. 6,080,725; 5,977,081, and5,443,829, each of which is incorporated herein in its entirety. The useof modified saponins adapted for, e.g., drug delivery, such as thosedescribed in U.S. Pat. Nos. 5,650,398; 5,443,829 and 5,273,965, each ofwhich is incorporated herein in its entirety, is also encompassed by theinvention. Preferably, the saponin is a single saponin. In otherembodiments of the invention, the term “saponin” covers mixtures ofsaponins. Suitable saponins include QS-7, QS-17, QS-18 and QS-21.Preferably, the mixture of saponins comprises two or more substantiallypure saponins. More preferably, the two or more substantially puresaponins are from Quillaja saponaria in doses that are otherwisesuboptimal for the individual saponins. In a particularly preferredembodiment, the combination of saponins consists essentially of twosubstantially pure saponins QS-7 and QS-21 or, in other particularlypreferred embodiments, QS-7 and QS-21-V1 or QS-7 and QS-21-V2, asdescribed in U.S. Pat. No. 6,231,859, which is herein incorporated byreference in its entirety. As used herein, “substantially pure” meanssubstantially free from compounds normally associated with the saponinin its natural state and exhibiting constant and reproduciblechromatographic response, elution profiles, and biologic activity. Theterm “substantially pure” is not meant to exclude artificial orsynthetic mixtures of the saponin with other compounds. A number ofnon-limiting examples of saponins and their methods of preparation areprovided below.

Saponins suitable for use in an individual are soluble in aqueoussolution and can be reconstituted from lyophilized or dried saponins.Specific saponins useful for the present invention include, but are notlimited to, the “Quil-A” adjuvant preparation sold by Superfos ofDenmark, and the chromatographic fractions with adjuvant activity thatare described in U.S. Pat. Nos. 5,057,540 and 5,583,112, particularlyfractions QS-21 (also referred to in the patents as QA-21) and QS-7.

Also useful in the methods and compositions of the present invention arechemically modified saponins that retain immune stimulating activity.According to Kensil et al., U.S. Pat. No. 5,583,112, the contents ofwhich are fully incorporated by reference herein, the carboxyl group onthe glucuronic acid of saponins from Quillaja saponaria Molina can beconjugated to a protein, a peptide, or a small molecule containing aprimary amine. According to Higuchi et al., 1987, Phytochemistry 26:229,saponins from Quillaja saponaria may be deacylated by alkaline-catalyzedhydrolysis. According to Marciani et al., U.S. Pat. No. 5,977,081, thecontents of which are fully incorporated by reference herein, thecarboxyl group on the glucuronic acid of nonacylated or deacylatedsaponins from Quillaja saponaria may be conjugated to a lipid, fattyacid, polyethylene glycol, or terpene.

Alternatively, an active fragment or synthetically modified derivativeof a fragment or a native saponin(s) may be utilized, such as thosedescribed in Soltysik et al., 1995, Vaccine 13(15):1403-1410; Marcianiet al., 2000, Vaccine 18:3141-3151. Such modifications include but arenot limited to removals or substitutions of saccharide residues,addition of saccharide residues, and removal, substitution and/oraddition of acyl chains.

The methods and compositions of the present invention may also employsaponins isolated from plant species other than Quillaja, such asGypsophila or Saponaria officinalis (Bomford et al, 1992, Vaccine10(9):572-577, incorporated by reference herein in its entirety), andChenopodium quinoa saponins (Estrada et al., 1998, Comp. Immunol.Microbiol. Infect. Dis. 21(3):225-36, incorporated by reference hereinin its entirety).

In certain embodiments of the invention, compositions of the inventioncomprises saponins in combination with excipients. Preferably, thesaponin is QS-21 and the excipients are selected from nonionicsurfactants, polyvinyl pyrolidone, human serum albumin, and variousunmodified and derivatized cyclodextrins. More preferably, in theseembodiments, the nonionic surfactants are selected from Polysorbate 20,Polysorbate-40, Polysorbate-60, and Polysorbate-80. The polyvinylpyrolidone may preferably be Plasdone C15, a pharmaceutical grade ofpolyvinyl pyrolidone. Preferred cyclodextrins arehydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, andmethyl-β-cyclodextrin. Preferably, the cyclodextrins areβ-cyclodextrins. Examples of the excipients include those described inPCT/US98/17940, incorporated by reference herein in its entirety.

In another embodiment of the invention, compositions of the inventioncomprises saponins in combination with immunostimulatory polymers.Preferably, the saponin is QS-21 and the immunostimulatory polymers areselected from cytokines, muramyl dipeptides and tripeptide derivatives,CpG dinucleotides, CpG oligonucleotides, monophosphoryl Lipid A, andpolyphosphazenes. Examples of the immunostimulatory polymers includethose described in PCT/US00/23688.

There are multiple acceptable techniques for extraction and isolation ofsaponins from Quillaja saponaria Molina bark. Acceptable procedures forpurifying the saponins of the present invention from Quillaja saponariaMolina bark, measuring the saponins for immune adjuvant activity, andcharacterizing the substantially pure saponins are disclosed in U.S.Pat. Nos. 5,057,540 and 5,583,112.

Aqueous extracts of Quillaja saponaria bark are also availablecommercially. These are dark brown, foamy extracts that contain manycompounds (tannins, polyphenolics, saponins) that can be analyzed by amethod such as reversed phase BPLC.

An example of a reversed phase HPLC analysis of a typical bark extractthat is suitable for purification of saponins is shown in FIG. 1 of U.S.Pat. No. 6,231,859, which is incorporated herein in its entirety.

Partial purification to enrich the saponin fraction and to remove themajority of tannins and polyphenolics can be accomplished by dialysis ofthe extract against water through a 10,000 molecular weight membrane orultrafiltration. The saponin fraction is retained.

Alternatively, an aqueous saponin extract can be pretreated withpolyvinylpolypyrrolidone to remove high molecular weight tannins andpolyphenolics through absorption of these compounds.

Residual tannins and polyphenolics can then be removed from the saponinfraction by diafiltration against water. The saponin fraction, whichforms micelles, is retained by ultrafiltration membranes of 10,000 to30,000 molecular weight cutoff pore size. This yields a partiallypurified extract that consists predominantly of diverse saponins.

Separation of saponins can be accomplished by chromatography in organicsolvents or organic solvent/water mixtures. A separation of saponins onsilica was described in U.S. Pat. No. 5,057,540. This yields saponins ofintermediate purity (enriched in an individual saponin, but less thansubstantially pure).

Alternatively, other solvent systems on silica gel or the use of reversephase chromatography can be used to accomplish the initial separation ofsaponins. This initial purification step can then typically be followedby reversed phase chromatography or similar HPLC step to purify thesaponins to near homogeneity.

For example, saponin extract may be recovered from plant cell materialfreshly extracted from Quillaja trees. Dialyzed extract is then purifiedon an ion exchange column, e.g., the DE-52 type, followed by SephadexG50 gel filtration. Ultrafiltration may be used instead of gelfiltration. The purified saponin composition is then subjected toRP-HPLC analysis on a VYDAC C4 column, eluted with 30-45% acetonitrilein a 0.15% aqueous TFA-solution.

The substantially pure saponins useful in the present invention may alsobe isolated from fresh plant material consisting of substantially livingcells as disclosed in WO 95/09179, or the previously describedprocedures.

The same procedure may be performed on plant cell material obtained bymeans of tissue culture or suspension cell culture. See, e.g., U.S. Pat.No. 5,716,848, which is incorporated herein by reference in itsentirety.

General guidance on the use of saponins, Quil-A, and QS-21 can be foundin the referenced patents. The amount of saponin present in apharmaceutically effective composition should contain about 0.1 to 5,000micrograms or more of a saponin. The amount of saponin present in apharmaceutically effective composition is more preferably from about 1to about 1000 micrograms, more preferably from about 5 to 500micrograms, and most preferably from about 10 to 100 micrograms. Incertain specific embodiments, the amount of saponin present in apharmaceutical composition of the invention is 1, 2, 3, 5, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 125 or 150 micrograms.

Formulations of pharmaceutical compositions comprising saponins andprocedures for their manufacture can be found in the literature and inthe U.S. patents incorporated by reference into this Description.Saponin formulations referred to are provided herein as nonlimitingexamples. In U.S. Pat. No. 5,583,112 at column 22, lines 11-17, apharmaceutically effective composition for intradermal administrationwas made by reconstituting lyophilized “Quil A”, a crude saponinmixture, into a phosphate buffered saline (PBS) solution and mixed witha solution containing 10 micrograms of Bovine Serum Albumin (BSA) toachieve a final volume of 200 microliters for intradermal injection. Theeffective amount of “Quil A” was found to be approximately 30-77micrograms of “Quil A” by dry weight.

In U.S. Pat. No. 5,583,112 at column 23, lines 35-38, a pharmaceuticallyeffective composition for subcutaneous administration was made by mixinga stock saline solution of ovalbumin and 10 micrograms of QS-21.

Also in U.S. Pat. No. 5,583,112 at Column 23, lines 35-38, apharmaceutically effective composition for subcutaneous administrationwas made by chemically crosslinking QS-21 to lysozyme as described inExample 18 of the patent and resuspending lyophilized QS-21/lysozymeconjugate into 200 microliters of PBS (pH 7) for a final concentrationof 10 micrograms of lysozyme and 1.6 micrograms of QS-21.

In a paper by Wu et al. (1994, Cellular Immunology 154:393-406), apharmaceutically effective composition for subcutaneous orintraperitoneal administration is disclosed containing 25 microgramsovalbumin absorbed to 250 micrograms of Al(OH)₃ and 20 micrograms ofQS-21 per immunization dose.

In another paper by Wu et al (1994, J. Immun. 148(5):1519-1525), apharmaceutically effective composition for immunization is disclosedcontaining 25 micrograms of a truncated recombinant HIV-1 envelopeprotein absorbed to 250 micrograms of Al(OH)₃ and 10 micrograms of QS-21in a sterile saline per immunization dose.

As a last non-limiting example, a pharmaceutically effective vaccine hasrecently been tested in human patients containing 5-500 micrograms of asynthetic nonapeptide and 100 micrograms of QS-21 in 500 microliters PBS(pH 7.4) per intradermally-administered dose (Lewis et al., 2000, Int.J. Cancer 87(3):391-398).

The optimum amount of a specific saponin for use with a specificcomposition of the invention may vary. Optimization of the specificsaponin amount for a given composition is, as demonstrated by theexamples cited above, well within the purview of the skilled artisan.

4.3. Compositions of the Invention and Uses Thereof

The present invention encompasses therapies which involve administeringan immunoreactive reagent and a saponin to an animal, preferably amammal, and most preferably a human, for preventing, treating, orameliorating symptoms associated with a disease, disorder, or infection.Prophylactic and therapeutic compounds of the invention include, but arenot limited to, immunoreactive reagents and a saponin. Immunoreactivereagents such as antibodies may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein. Thecompositions of the invention can also be used in conjunction with otherforms of therapy for a particular disease.

Compositions of the present invention can be administered to an animal,preferably a mammal and most preferably a human, to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection. In a preferred embodiment, the composition of the inventionexists outside of the body. Preferably, the immunoreactive reagent ofthe invention has been established to have some therapeutic benefit inthe absence of saponin, and recognizes an epitope on a cell or moleculeassociated with the cause or symptoms of a disease, disorder orinfection.

For example, the compositions and methods of the invention can also beused to prevent, inhibit or reduce the growth or metastasis of cancerouscells. In a specific embodiment, a composition comprising animmunoreactive reagent, such as an antibody, and saponin inhibits orreduces the growth or metastasis of cancerous cells by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the growth or metastasis in absence of saidcomposition.

Each composition of the invention should contain at least oneimmunoreactive reagent (as defined herein, e.g., an antibody) and asaponin, and can then further comprise other reagents such as cytokines,growth factors, immunostimulatory oligonucleotides, and the like. One ormore immunoreactive reagents that immunospecifically bind to one or moretarget antigens may be used locally or systemically in the body as atherapeutic. The immunoreactive reagents may also be advantageouslyutilized in combination with other such reagents such as monoclonal orchimeric antibodies, or with immune active compounds such as lymphokinesor hematopoietic growth factors, such as, e.g., IL-2, IL-3 and IL-7 orimmune response modifiers (IRMs, 3M Pharmaceuticals, St. Paul, Minn.),which, for example, serve to increase the number and/or activity ofimmune effector cells which act in conjunction with the immunoreactivereagent. In addition, immunostimulatory oligonucleotides may be used incombination with the saponin and immunoreactive reagents. Sucholigonucleotides are known to enhance the immune response. Woolridge, etal., 1997, Blood 89:2994-2998. Such oligonucleotides are described inInternational Patent Publication Nos. WO 01/22972, WO 01/51083, WO98/40100 and WO 99/61056, each of which is incorporated herein in itsentirety, as well as U.S. Pat. Nos. 6,207,646 and 6,194,388, each ofwhich is incorporated herein in its entirety. Such immunostimulatoryoligonucleotides can comprise an unmethylated CpG motif. Other kinds ofimmunostimulatory oligos such as phosphorothioate oligodeoxynucleotidescontaining YpG- and CpR-motifs have been described by Kandimalla et al.in “Effect of Chemical Modifications of Cytosine and Guanine in aCpG-Motif of Oligonucleotides: Structure-Immunostimulatory ActivityRelationships.” Bioorganic & Medicinal Chemistry 9:807-813 (2001),incorporated herein by reference in its entirety. Methods of determiningthe activity of such oligonucleotides can be performed as described inthe aforementioned patents and publications. Moreover, immunostimulatoryoligonucleotides can be modified within the phosphate backbone, sugar,nucleobase and internucleotide linkages in order to modulate theactivity. Such modifications are known to those of skill in the art.

The immunoreactive reagents and saponins of this invention may also beadvantageously utilized in combination with one or more drugs used totreat a disease, disorder, or infection such as, for example anti-canceragents, anti-inflammatory agents, or anti-bacterial/fungal or anti-viralagents. Examples of anti-cancer agents include, but are not limited to,cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors(e.g., CPT-11, topotecan, 9-ANTIGENIC COMPOSITION, and GG-211),gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU),leucovorin, vinorelbine, temodal, and taxol.

In a specific embodiment, immunoreactive reagents administered to ananimal are of a species origin or species reactivity that is the samespecies as that of the animal. Thus, in a preferred embodiment, human orhumanized antibodies are administered to a human patient for therapy orprophylaxis.

The invention provides methods of treatment, prophylaxis, andamelioration of one or more symptoms associated with a disease, disorderor infection by administering to a subject of an effective amount of animmunoreactive reagent and a saponin, or pharmaceutical compositioncomprising an immunoreactive reagent and a saponin. In a preferredaspect, the immunoreactive reagent and saponin are substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). In a specific embodiment, thesubject is an animal, preferably a mammal such as non-primate (e.g.,cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkeysuch as a cynomolgous monkey and a human). In a preferred embodiment,the subject is a human.

Various delivery systems are known and can be used to administer animmunoreactive reagent, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe immunoreactive reagent, etc. Methods of administering animmunoreactive reagent and saponin or a pharmaceutical compositioncomprising the same include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intraperitoneal,intravenous and subcutaneous), epidural, and mucosal (e.g., intranasaland oral routes). Preferably, the immunoreactive reagent is administeredintravenously, while the saponin is not administered intravenously. In aspecific embodiment, immunoreactive reagents, for example, antibodies,are administered intramuscularly, intravenously, or subcutaneously. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety. In apreferred embodiment, an immunoreactive reagent is administered usingAlkermes AIR™ pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.). Preferably, the saponin is not delivered by pulmonaryadministration.

In accordance with the present invention, a composition of theinvention, comprising an immunoreactive reagent and a saponin isadministered to a human subject with cancer, an infectious disease, or aneurodegenerative or amyloid diseases as a treatment. In one embodiment,“treatment” or “treating” refers to an amelioration of cancer, aninfectious disease, or a neurodegenerative or amyloid disease, or atleast one discernible symptom thereof. In another embodiment,“treatment” or “treating” refers to an amelioration of at least onemeasurable physical parameter associated with cancer, an infectiousdisease, a neurodegenerative or amyloid disease, notnecessarily-discernible by the subject. In yet another embodiment,“treatment” or “treating” refers to inhibiting the progression of acancer, an infectious disease, a neurodegenerative or amyloid disease,either physically, e.g., stabilization of a discernible symptom,physiologically, e.g., stabilization of a physical parameter, or both.In yet another embodiment, “treatment” or “treating” refers to delayingthe onset of a cancer, a neurodegenerative or amyloid disease.

In certain embodiments, the compositions of the present invention areadministered to a human subject as a preventative measure against suchcancer, an infectious disease, a neurodegenerative or amyloid disease.As used herein, “prevention” or “preventing” refers to a reduction ofthe risk of acquiring a given cancer, infectious disease,neurodegenerative or amyloid disease. In one mode of the embodiment, thecompositions of the present invention are administered as a preventativemeasure to a human subject having a genetic predisposition to a cancer,infectious disease, neurodegenerative or amyloid disease. In anothermode of the embodiment, the compositions of the present invention areadministered as a preventative measure to a subject having a non-geneticpredisposition to a cancer, or to a subject facing exposure to an agentof an infectious disease.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment or prevention. In one embodiment, the treatment orprevention may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,care is taken to use materials to which the immunoreactive reagent doesnot absorb. In a particular embodiment, the immunoreactive reagent isadministered systemically, for example, by i.v., and the saponin isadministered locally to the area in need of treatment or prevention.

In another embodiment, the composition can be delivered in a vesicle, inparticular a liposome (see Langer, Science, 249:1527-1533, 1990; Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the composition can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies, or one or more fusionproteins. See, e.g., U.S. Pat. No. 4,526,938; PCT publication WO91/05548; PCT publication WO 96/20698; Ning et al., “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology, 39:179-189, 1996; Songet al., “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions,” PDA Journal of Pharmaceutical Science & Technology,50:372-397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for abFGF Antibody for Cardiovascular Application,” Pro. Intl. Symp. Control.Rel. Bioact. Mater., 24:853-854, 1997; and Lam et al.,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.,24:759-760, 1997, each of which is incorporated herein by reference inits entirety. In one embodiment, a pump may be used in a controlledrelease system (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; and Saudek et al.,1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used to achieve controlled release of immunoreactivereagents (see e.g., Medical Applications of Controlled Release, Langerand Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597;U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No.5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO99/20253). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target (e.g., the lungs), thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer,1990, Science 249:1527-1533).

The invention also provides that an immunoreactive reagent, for examplean antibody, is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of immunoreactive reagent.In one embodiment, the immunoreactive reagent and saponin are suppliedtogether or separately as dry sterilized lyophilized powders or waterfree concentrates in one or more hermetically sealed containers and canbe reconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. The effective dosage ofeach immunoreactive reagent, and can be estimated initially from invitro assays. It also depends on the nature of the target antigen, thedensity of the antigen in the tumors, the tumor type, the manner ofadministration, which can be optimized by a person skilled in the artwithout undue experimentation. Usual effective dosages for injectionrange from about 0.1 to 5 mg/kg/day, preferably from about 1 to 4mg/kg/day, and more preferably from 2 to 4 mg/kg/week. Preferably, theimmunoreactive reagent is supplied as a dry sterile lyophilized powderin a hermetically sealed container at a unit dosage of at least 5 mg,more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least35 mg, at least 45 mg, at least 50 mg, or at least 75 mg.

In other embodiments, the saponin is QS-7, QS-17, QS-18, QS-21,QS-21-V1, or QS-21-V2. Preferably, the saponin is supplied at a unitdosage of at least 1 microgram, more preferably at least 10 micrograms,at least 15 micrograms, at least 25 micrograms, at least 35 micrograms,at least 45 micrograms, at least 50 micrograms, or at least 75micrograms. In a preferred embodiment, the amount of saponin in thepharmaceutical composition is from about 0.1 to about 1000 micrograms.In a preferred embodiment, the amount of saponin is about 1, 2, 3, 5,10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 125, 150, 200,250, 300 or 500 micrograms. In a particularly preferred embodiment, theamount of saponin is 100 micrograms. In a particularly preferredembodiment, the saponin is QS-21. The amount of QS-21 in thepharmaceutical compositions is preferably about 1 microgram or more. Ina particularly preferred embodiment, the amount of QS-21 is from about10 to about 1000 micrograms. In a particularly preferred embodiment, theamount of QS-21 is about 10 to 100 micrograms, or 20 to 50 micrograms,preferably 25 or 50 micrograms. The lyophilized immunoreactive reagentand saponin should be stored at between 2 and 8° C. in its originalcontainer and should be administered within 12 hours, preferably within6 hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, an immunoreactive reagentand saponin are supplied in liquid form in a hermetically sealedcontainer indicating the quantity and concentration of the saponin andimmunoreactive reagent. Preferably, the liquid form of theimmunoreactive reagent is supplied in a hermetically sealed container atleast 1 mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, atleast 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, or at least 25mg/ml. Preferably, the liquid form of the saponin is supplied in ahermetically sealed container at least 1 mg/ml, more preferably at least2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, atleast 15 mg/mil, or at least 25 mg/ml.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of an immunoreactive reagent and a saponin, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent, adjuvant(e.g., MPL, immunostimulatory oligonucleotides, Freund's complete andincomplete, mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful adjuvants for humans such as BCG (BacilleCalmette-Guerin) and Corynebacterium parvum), excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents: Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa prophylactically or therapeutically effective amount of theimmunoreactive reagent and saponin, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Preferably, the saponin is not administeredintravenously.

Generally, the ingredients of compositions of the invention are suppliedas a kit either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration. In another embodiment, a kit of theinvention further comprises a needle or syringe, preferably packaged insterile form, for injecting the composition, and/or a packaged alcoholpad. Instructions are optionally included for administration of thecompositions of the invention by a clinician or by the patient.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disease, disorder, or infection can be determined bystandard clinical techniques. The precise dose to be employed in theformulation will depend on the route of administration, the age of thesubject, and the seriousness of the disease, disorder, or infection, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model (e.g., thecotton rat or Cynomolgous monkey) test systems. Models and methods forevaluation of the effects of saponins and antibodies, or otherimmunoreactive reagents are known in the art. (Wooldridge et al., Blood,1997, 89 (8): 2994-2998, incorporated by reference herein in itsentirety).

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage will, however, dependupon the extent to which the serum half-life of the molecule has beenincreased. Generally, human antibodies have longer half-lives within thehuman body than antibodies from other species due to the immune responseto the foreign polypeptides. Thus, lower dosages of human antibodies andless frequent administration is often possible. Further, the dosage andfrequency of administration of immunoreactive reagents may be reducedalso by enhancing uptake and tissue penetration (e.g., into the lung) ofthe immunoreactive reagents such as, for example, lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of an immunoreactive reagent and saponin can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with an immunoreactive reagentin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In a preferred example, a subject is treatedwith a saponin in the range of between about 0.1 to 30 mg/kg bodyweight, one time per week for between about 1 to 10 weeks, preferablybetween 2 to 8 weeks, more preferably between about 3 to 7 weeks, andeven more preferably for about 4, 5, or 6 weeks. In other embodiments,the pharmaceutical composition of the invention is administered once aday, twice a day, or three times a day. In other embodiments, thepharmaceutical composition is administered once a week, twice a week,once every two weeks, once a month, once every six weeks, once every twomonths, twice a year or once per year. It will also be appreciated thatthe effective dosage of the immunoreactive reagents used for treatmentmay increase or decrease over the course of a particular treatment. Theimmunoreactive reagent and the saponin can be administeredsimultaneously. In a preferred embodiment, the saponin andimmunoreactive reagent are administered at different times. In oneembodiment, the immunoreactive reagent is administered prior to thesaponin, e.g., at least 7 days, 3 days, 2 days, 1 day, 16 hours, 12hours, 8 hours, 4 hours, 2 hours or 1 hour prior. In a preferredembodiment, the saponin is administered prior to the immunoreactivereagent, e.g., at least 7 days, 3 days, 2 days, 1 day, 16 hours, 12hours, 8 hours, 4 hours, 2 hours or 1 hour prior.

4.4. Methods of Making Pharmaceutical Compositions Comprising anImmunoreactive Reagent and a Saponin

The present invention encompasses methods of making pharmaceuticalcompositions comprising a saponin and an immunoreactive reagent.

In one embodiment, the saponin is combined with the immunoreactivereagent. In a preferred embodiment, the saponin and immunoreactivereagent are formulated separately, and are in separate containers withina kit. Such a kit can optionally further comprise instructions for theadministration of the immunoreactive reagent and saponin.

5. EXAMPLES

5.1. Enhancement of Antibody Mediated Lysis In Vitro

Murine splenocytes (effector cells) are generated from the spleens ofnaive 6-8 week old mice. These effector cells are incubated with 1 to 10ug/ml of QS-21 or appropriate amount of another immunomodulatory saponinfor 24 to 72 hours. At the end of the incubation period, target cells(E.G7-OVA or MO4) are loaded with 51Cr. Effector cells and labeledtarget cells are incubated at defined effector:target ratios in thepresence and absence of monoclonal antibody to SIINFEKL/Class I MHC (1to 10 ug/ml) at 4° C. for 30 to 60 minutes. The lysis in the presence ofQS-21 and monoclonal is compared to controls without QS-21, withoutmonoclonal, or both. There is an enhanced lysis due to QS-21 (determinedby the fold-enhancement of lysis due to QS-21/monoclonal over monoclonalalone).

5.2. Improvement of Protection in Tumor Challenge Model

C57B1/6 mice are inoculated by s.c. route in the flank with M04 tumor(1×10⁵) or EG7-OVA tumor. At 24 to 48 hours after inoculation, mice areinjected by IP route with a monoclonal antibody to SIINFEKL/Class I MHC,or by local SC route in the presence or absence of QS-21 (10-20 ug). Theantitumor effect of the QS-21/monoclonal antibody treatment is comparedto that of monoclonal antibody treatment by monitoring the growth of thetumors over a 30 to 60 day period (measurement with calipers). Survivalwas determined, and significance with respect to time to death wasassessed using Cox regression analysis. Mice were also observed dailyfor signs of toxicity including level of activity, ruffled fur,diarrhea, and general appearance. A significant number of mice treatedwith monoclonal antibody alone developed tumor compared with thosetreated with antibody and QS-21. The benefit of the QS-21 treatment isdemonstrated by a delay in tumor progression. Models and methods forevaluation of the effects of saponins and antibodies, or otherimmunoreactive reagents are known in the art. (Wooldridge et al., Blood,1997, 89 (8): 2994-2998, incorporated by reference herein in itsentirety).

5.3. Improved Opsonization of Bacteria by Use of OS-21

Improved opsonization of bacteria by saponin is demonstrated in vitro byincubating effector cells for the opsonophagocytosis assay (HL-60) withQS-21. The cells are evaluated for whether they are more effective inopsonizing S. pneumonia or S. aureus at a given antibody titer (forexample a human serum sample with opsonizing activity specific for S.pneumonia or S. aureus, respectively).

5.4. The Upregulation by QS-21 of Fc Receptors

Monocytes, natural killer cells, or polymorphonuclear cells areincubated in the presence or absence of QS-21 (for example 1-10 ug/ml).The trypsinized cells are incubated at 4° C. for 60 min with monoclonalantibodies specific to Fc a R, Fc gamma R1, Fc gamma RII, or Fc gammaRIII. The cells are then incubated with an anti-mouse IgG FITC probe,washed, fixed in parafomaldehyde, and analyzed by FACScan. Upregulationof Fc receptors on these cells is demonstrated.

In addition, upregulation of TNF-alpha, IL-6 and MIP-1-alpha by QS-21 inmacrophage cells was demonstrated. In bone marrow derived dendriticcells, QS-21 was found to increase production of MIP-1-alpha and IL-1,and to decrease the production of Il-12 and MIP-1-beta.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of each of whichare incorporated by reference herein in their entireties.

1. A pharmaceutical composition comprising a saponin and animmunoreactive reagent, wherein said immunoreactive reagent specificallybinds an antigen selected from the group consisting of atumor-associated antigen, an antigen of an agent of infectious disease,an antigen associated with a neurodegenerative disease, and an antigenassociated with an amyloid disease.
 2. The pharmaceutical composition ofclaim 1, wherein the immunoreactive reagent is an antibody.
 3. Thepharmaceutical composition of claim 2, wherein the amount of saponin isat least 1 microgram.
 4. The pharmaceutical composition of claim 3,wherein the amount of saponin is 10 to 20 micrograms.
 5. Thepharmaceutical composition of claim 3, wherein the amount of saponin is20 to 100 micrograms.
 6. The pharmaceutical composition of claim 3,wherein the amount of saponin is 100 to 500 micrograms.
 7. Thepharmaceutical composition of claim 1, wherein the saponin is QS-7,QS-17, QS-18, QS-21, QS-21-V1, or QS-21-V2.
 8. The pharmaceuticalcomposition of claim 7, wherein the saponin is QS-21.
 9. Thepharmaceutical composition of claim 1, wherein the immunoreactivereagent specifically binds a tumor-associated antigen.
 10. Thepharmaceutical composition of claim 9, wherein the tumor is anon-Hodgkin's lymphoma, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, polycythemia vera, lymphoma, multiple myeloma,Waldenström's macroglobulinemia, or heavy chain disease (B-celllymphoma).
 11. The pharmaceutical composition of claim 9, wherein thetumor-associated antigen is Melan A, MART-1, MAGE-1, MAGE-3, BAGE,GAGE-1, GAGE-2, tyrosinase, gp100, gp75, HER-2/neu, c-erb-B2, CEA, PSA,MUC-1, CA-125, Stn, TAG-72, KSA (17-1A), PSMA, p53, RAS, EGF-R, VEGF,GD2, GM2, GD3, Anti-Id, CD20, CD19, CD22, CD36, Aberrant class II, B1,CD25, or BPV.
 12. The pharmaceutical composition of claim 1, wherein theimmunoreactive reagent binds specifically to an antigen of an agent ofinfectious disease.
 13. The pharmaceutical composition of claim 12,wherein the infectious disease is a viral disease.
 14. Thepharmaceutical composition of claim 13, wherein the viral disease isviral meningitis, encephalitis, dengue or smallpox, or is a viraldisease caused by hepatitis type A, hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type U (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and Epstein Barr virus.
 15. The pharmaceutical compositionof claim 1, wherein the infectious disease is a bacterial disease. 16.The pharmaceutical composition of claim 15, wherein the bacterialdisease is caused by infection by mycobacteria rickettsia, mycoplasma,neisseria, streptococcus, staphylococcus, mycobacterium, tetanus,pertissus, anthrax, cholera, plague, diptheria, or chlamydia.
 17. Thepharmaceutical composition of claim 1, wherein the infectious disease isa protozoal disease.
 18. The pharmaceutical composition of claim 17,wherein the protozoa is leishmania, kokzidioa, trypanosoma, or malaria.19. The pharmaceutical composition of claim 1, wherein theimmunoreactive reagent binds specifically to an antigen associated witha neurodegenerative or amyloid disease.
 20. The pharmaceuticalcomposition of claim 19, wherein the neurodegenerative or amyloiddisease is Alzheimer's Disease, age-related loss of cognitive function,senile dementia, Parkinson's disease, amyotrophic lateral sclerosis,Wilson's Disease, cerebral palsy, progressive supranuclear palsy, Guamdisease, Lewy body dementia, prion diseases, spongiformencephalopathies, Creutzfeldt-Jakob disease, polyglutamine diseases,Huntington's disease, myotonic dystrophy, Freidrich's ataxia, ataxia,Gilles de la Tourette's syndrome, seizure disorders, epilepsy, chronicseizure disorder, stroke, brain trauma, spinal cord trauma, AIDSdementia, alcoholism, autism, retinal ischemia, glaucoma, autonomicfunction disorder, hypertension, neuropsychiatric disorder,schizophrenia, or schizoaffective disorder, type II diabetes,amyloidoses associated with chronic inflammatory disease, amyloidosesassociated with infectious disease, or myeloma.
 21. The pharmaceuticalcomposition of claim 20, wherein the antigen is β-amyloid, an oligomericAβ complex, an ApoE4-Aβ complex, tau protein, a mutant amyloidprecursor, a mutant of presenillin, α-synuclein, a prion protein, or anantigenic fragment of any of the foregoing proteins.
 22. A method oftreating or preventing cancer in an individual in whom such treatment orprevention is desired, comprising administering to the individual anamount of an immunoreactive reagent that specifically binds to anantigen of said type of cancer, and a saponin, which amount is effectiveto treat or prevent cancer in the individual.
 23. A method of treatingor preventing an infectious disease in an individual in whom suchtreatment or prevention is desired, comprising administering to theindividual an amount of an immunoreactive reagent that specificallybinds to an antigen of said type of cancer, and a saponin, which amountis effective to treat or prevent the infectious disease in theindividual.
 24. A method of treating or preventing a neurodegenerativeor amyloid disease in an individual in whom such treatment or preventionis desired, comprising administering to the individual an immunoreactivereagent that specifically binds to an antigen of said neurodegenerativeor amyloid disease, and a saponin, which amount is effective to treat orprevent the neurodegenerative or amyloid disease in the individual. 25.The method of claim 22, 23 or 24, wherein the immunoreactive reagent isan antibody.
 26. The method of claim 22, 23 or 24, wherein the amount ofsaponin is at least 1 microgram.
 27. The method of claim 26, wherein theamount of saponin is 10 to 20 micrograms.
 28. The method of claim 27,wherein the amount of saponin is 20 to 100 micrograms.
 29. The method ofclaim 27, wherein the amount of saponin is 100 to 500 micrograms. 30.The method of claim 22, 23 or 24, wherein the saponin is QS-7, QS-17,QS-18, QS-21, QS-21-V1, or QS-21-V2.
 31. The method of claim 30, whereinthe saponin is QS-21.
 32. A method of enhancing antibody dependentcellular cytotoxicity or phagocytosis of a target in an individual inwhom such enhancement is desired, comprising administering to theindividual an amount of an immunoreactive reagent that specificallybinds to an antigen of said target, and a saponin, which amount iseffective to enhance antibody dependent cellular cytotoxicity orphagocytosis of the target in the individual.
 33. The method of claim32, wherein the immunoreactive reagent is an antibody.
 34. The method ofclaim 32, wherein the amount of saponin is at least 1 microgram.
 35. Themethod of claim 34, wherein the amount of saponin is 10 to 20micrograms.
 36. The method of claim 34, wherein the amount of saponin is20 to 100 micrograms.
 37. The method of claim 34, wherein the amount ofsaponin is 100 to 500 micrograms.
 38. The method of claim 32, whereinthe saponin is QS-7, QS-17, QS-18, QS-21, QS-21-V1, or QS-21-V2.
 39. Themethod of claim 38, wherein the saponin is QS-21.
 40. A method ofenhancing passive immunotherapy in an individual in whom suchenhancement is desired, wherein said immunotherapy comprisesadministration of an amount of an immunoreactive reagent thatspecifically binds to an antigen of a target, said method comprisingfurther administering an amount of a saponin, which amount is effectiveto enhance the passive immunotherapy in the individual.
 41. The methodof claim 40, wherein the immunoreactive reagent is an antibody.
 42. Themethod of claim 40, wherein the amount of saponin is at least 1microgram.
 43. The method of claim 42, wherein the amount of saponin is10 to 20 micrograms.
 44. The method of claim 42, wherein the amount ofsaponin is 20 to 100 micrograms.
 45. The method of claim 42, wherein theamount of saponin is 100 to 500 micrograms.
 46. The method of claim 40,wherein the saponin is QS-7, QS-17, QS-18, QS-21, QS-21-V1, or QS-21-V2.47. The method of claim 46, wherein the saponin is QS-21.
 48. A kitcomprising, in one or more containers, a saponin and an immunoreactivereagent, wherein said immunoreactive reagent specifically binds anantigen selected from the group consisting of a tumor-associatedantigen, an antigen of an agent of infectious disease, an antigenassociated with a neurodegenerative disease, and an antigen associatedwith an amyloid disease.
 49. The kit of claim 48, wherein the saponinand immunoreactive reagent are in separate containers.
 50. The kit ofclaim 49, wherein the immunoreactive reagent is formulated forintravenous administration, and the saponin is formulated foradministration other than intravenous administration.
 51. The kit ofclaim 49, wherein the immunoreactive reagent is an antibody.
 52. The kitof claim 49, wherein the saponin is QS-21.
 53. The kit of claim 49,wherein the immunoreactive reagent is an antibody and the saponin isQS-21.
 54. The method of claim 22, 23, 24, 32, or 40, wherein theimmunoreactive reagent and the saponin are administered at differenttimes from one another.
 55. The method of claim 54, wherein the saponinis administered prior to the immunoreactive reagent.
 56. The method ofclaim 55, wherein the saponin is administered at least one day prior tothe immunoreactive reagent.